J. A@c. Food Chem. 1990, 38, 1595-1600
1595
Photoinduced Oxidation of the Organophosphorus Fungicide Tolclofos-methyl on Clay Minerals Toshiyuki Katagi Biochemistry and Toxicology Laboratory, Sumitomo Chemical Co., Ltd., 4-2-1 Takatsukasa, Takarazuka, Hyogo 665,Japan
Photodegradation of the organophosphorus fungicide tolclofos-methyl (I) [0-(2,6-dichlor0-4methylphenyl) 0,O-dimethyl phosphorothioate] on kaolinite and montmorillonite was examined by using a 500-Wxenon lamp. Spectroscopic and photodegradation studies showed that photoinduced oxidation of I was the dominant degradation pathway on the clay minerals. The reactions of I with hydroxyl and hydroperoxyl radicals, which were formed by irradiation on the clay minerals in the presence of both molecular oxygen and water, were suggested as the main pathway in forming the oxon derivative
of I.
INTRODUCTION Photodegradation on the soil surface is one of the important factors that determines the environmental fate of pesticides (Miller and Crosby, 1983). Photodegradation studies using sunlight (Mikami et al., 1984;Smith et al., 1978) or artificial light (Allmain and Schmid, 1985; Burkhard and Guth, 1979)have revealed that the organophosphorus pesticides are mainly photodegraded via ester cleavage and/or P=S oxidation. Mikami et al. (1984) have reported that I is photodegraded on intact soils much faster than in water and that the main degradation pathways are P=S oxidation to the corresponding oxon derivative and demethylation of the 0-methyl group. Spencer et al. (1980),in their studies of the photodegradation of parathion, reported that clay minerals significantly affect the rate of photolysis and the amount of paraoxon formed. The objectives of the work described in this paper are, first, to determine whether adsorption of I onto clay minerals plays an important role in its photodegradation and, second, t o determine whether secondary degradation by active oxygen species could play a role. Thus, the first part of the work is concerned with photodegradation of I adsorbed onto clay minerals under irradiation from a xenon lamp. T h e second part is concernedwith studies on the extent to which active oxygen species such as singlet oxygen (Gohre and Miller, 1983, 1985;Zepp et al., 19811,superoxide anion radical (Draper and Crosby, 1983;Calvert e t al., 1954),hydroxyl radical (Draper and Crosby, 1981;Tseng and Chang, 1975),and atomic oxygen (Hauser et al., 1951) are developed under irradiation and participate in the degradation of I. MATERIALS AND METHODS Chemicals. Tolclofos-methyl (I), 0-(2,6-dichlor0-4-methylphenyl) 0,O-dimethyl phosphate (II), 0-[2,6-dichlor0-4(hydroxymethy1)phenyll0,O-dimethyl phosphorothioate (111), 0-[2,6-dichloro-4-(hydroxymethyl)pheny1]0,O-dimethyl phosphate (IV), 2,6-dichloro-4-methylphenol(V),and 2,6-dichloro44hydroxymethyl)phenol (VI) were synthesized according to methods already reported (Mihara et al., 1981). 0-(2,6Dichloro-4-methylphenyl) 0-methyl S-methyl phosphorothioate (VII) was synthesized by treatment of 0-(2,6-dichlor0-4methylphenyl) 0-methyl hydrogen phosphorothioate, prepared from I (Mihara et al., 1981),with methyl iodide in acetone. 3,5002 1-856 119011438-1595$02.50/0
Dichloro-4-hydroxybenzoicacid (VIII) was purchased from Aldrich Chemical Co., Ltd. (Milwaukee). [14C]-Iuniformly labeled in the phenyl ring (149.0 mCi/g, radiochemical purity >99%) was synthesized in our laboratory (Yoshitake et al., 1978). Hz18O (97 atom %) was purchased from E. Merck (Montreal). For the spectrophotometric assay of a trace amount of hydrogen peroxide, leuco crystal violet (Tokyo Kasei, Tokyo) and horseradish peroxidase (EC 1.11.1.7) (Type IV, 280 units/mg of protein; Sigma, St. Louis) were used. 2,bDimethylfuran (DMF) and acetonylacetone were purchased from Aldrich. transDiacetylethylene was synthesized by the reported method (Armstrong and Robinson, 1934). Pure water of electrical conductivity 5.5 X 10-8 W/cm was supplied from a Puric Model R equipped with a G-10 filter (Organ0 Co., Ltd., Osaka). Kaolinite and montmorillonite clay minerals were purchased from Wako Co., Ltd. (Osaka). Spectroscopy. The ultraviolet (UV) absorption spectrum of I in 10% aqueous acetonitrile was determined with a Hitachi U-3210 spectrophotometer using a quartz cuvette (1-cm path length). Reflectance spectra of I adsorbed on a thin film of each clay mineral were obtained with the same spectrophotometer equipped with a 60 i.d. integrating sphere. Each clay mineral (10 g), suspended in pure water, was uniformly spread over a glass plate (20 X 20 cm) and the excess water evaporated at room temperature overnight. I in 1,2-dichloroethane was uniformly applied to a 3 X 3-cm square, the clay film was prepared as above at the rate of 0.12 mg/cm*, and the solvent was evaporated at room temperature. The infrared (IR) spectra of I in KBr and clay pellets were obtained with a Nicolet ZDX Fourier transform spectrometer according to the method reported by Rockley and Rockley (1987). The 5-mL aliquot of 0.5 M 1,2-dichloroethanesolution of I was gently stirred with 100 mg of each clay mineral in darkness overnight. The clay mineral was separated by centrifugation (3000 rpm for 10 min) and twice washed with 1,2dichloroethane to remove excess I. After drying at room temperature overnight, each clay mineral (10 mg) was well mixed with 300 mg of KBr for the preparation of a pellet. Mass spectra were measured in electron ionization (EI)mode at 70 eV with a Hitachi M-80 mass spectrometer equipped with a Hitachi M-003 data processing unit. The production of transdiacetylethylene, via the reaction of DMF with singlet oxygen, was examined by high-performance liquid chromatography (HPLC). A Hitachi 655A liquid chromatograph equipped with an ODS A-112 reverse-phase column (6 mm i.d. X 15 cm, Sumika Analytical Service Ltd., Osaka) was operated at a flow rate of 1 mL/min, using acetonitrile-water (3:l) as a mobile phase. trans-Diacetylethylene and DMF were detected by a Hi@ 1990 American Chemical Society
1596 J. Agric. Food Chem., Vol. 38,No. 7, 1990
Katagi
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Figure 1. Apparatus used in the photodegradation study. (1) 500-W xenon lamp; (2) UV filter with radiation cutoff of 300 nm and infrared-reflectingcoating; (3) quartz glass; (4) thermometer connected to a thermocouple; (5) Pyrex beaker; (6) photoreaction vessel; (7) clay mineral thin f h (8)cooled water; (9) constant temperature bath with a circulator;(10) 10%NaOH; (11)distilled water; (12) ethylene glycol; (13) 0.5 M NaOH.
A
Figure 2. Absorption and reflectance spectra of I and emission spectrum of a 500-W xenon lamp. (a) Absorption spectrum of I in 10% acetonitrile (1.06 X 10-4 M); (b) reflectance spectrum of I adsorbed on kaolinite (0.12 mg/cm2); (c) emission spectrum of a 500-W xenon lamp. tachi 655A-11 UV monitor at 225 nm (retention times 2.7 and 3.2 min, respectively). Radioassay. The radioactivity of 14C was quantified with a Packard 460CD liquid scintillation spectrometer (LSC),using 10 mL of Packard Emulsifier-scintillator 299. The radioactivity bound to the clay was quantified by combustingan aliquot (100200 mg) with a Packard 306 sample oxidizer prior to the LSC measurement. The recovery of 14C in a sample oxidizer was >95% when 13 mL of Packard Permafluor-V and 9 mL of Carbo-sorb were used. Photolysis Studies. The apparatus used is shown diagramatically in Figure 1. The clay film was prepared from a suspension of 1g of each clay mineral in 2 mL of pure water in the bottom of a Pyrex glass beaker (3.5 cm i.d. X 5.0 cm height). After the water had evaporated, the resulting clay film was uniformly treated with [1'c]-I (149 pCi, 1 mg) in 1,2-dichloroethane and then continuously irradiated with a 500-W xenon lamp (USIO, Model UIV-515OXl3,Tokyo). The emission spectrum of a xenon lamp with a cutoff of 320 nm and a maximum of 468 nm was obtained by a Licor Model LI-1800 photometer (Figure 2). Air, free of C02 and saturated with water vapor, was passed continuously over the irradiated samples at a flow rate of 100 mL/ min. It was then passed through two gas-washing bottles to trap volatile 1%. The first contained ethylene glycol (100 mL) and the second 0.5M NaOH (400 mL). The temperature at the clay surface was maintained at 25 f 1 OC by a continuous flow of cooled water below the beakers. The temperature was monitored by attaching to the clay surface a Chino C060-K thermo-
couple connected to a Chino CD700 thermometer. The dark control experiment was conducted in the same way except that the photoreaction vessel was covered with aluminum foil. At an appropriate interval, samples of the clay were scraped from the beaker in the photoreaction vessel and extracted with 30 mL of ethyl acetate followed by centrifugation at 3000 rpm for 10 min. After decantation, the extraction was repeated twice. A 0.5-mL aliquot of the combined supernatant liquid was radioassayed in duplicate and the remaining portion used for TLC analysis after being concentrated in a rotating evaporator. I and its degradation products were separated on TLC (6OFzM,20 X 20 cm, 0.25 mm layer thickness, E. Merck) with a solvent system of toluene-acetic acid (7:1), and the autoradiogram was prepared. The amount of each product was quantified by radioassay of the 14C gel region scraped from the TLC plate. Chemical identification was by cochromatographyof the extracts with the authentic standards. The unextractable 14C bound to the clay residue was radioassayed by a sample oxidizer and LSC. For the determination of volatile activity, the gas-washing bottles were sampled periodically. A 2.0-mL aliquot of ethylene glycol was radioassayed in duplicate, the remaining glycol was extracted twice with 100 mL of ethyl acetate, and the extract was analyzed by TLC. A 2.0-mL aliquot of sodium hydroxide was also radioassayed. In this case, a further aliquot (10 mL) of the remaining solution was treated with 20 mL of 1 M BaC12 to precipitate BaW03, which was separated by centrifugation. A 2.0-mL aliquot of the supernatant was radioassayed in duplicate to quantify 14C02. The liquid in the two traps was replaced at each sampling time. Separate experiments were conducted to examine the extent of indirect photolysis. The clay film treated with [%+I was irradiated under N2 (>99.99%) for 14 days to examine the effect of molecular oxygen. The dried clay film, prepared by heating at 120 "C for 2 h, was treated with [14C]-Iand irradiated in the same way under dry air to examine the effect of water. A further study on the photodegradation of [14C]-I on kaolinite prepared by using H21W (0.5 mL/g of clay) was conducted under air for 7 days to examine the incorporation of 180 into 11. The ratio of incorporation was estimated by MS analysis. Assay of Active Oxygen Species. The generation of hydrogen peroxide on the irradiated clays was examined as follows. Pure water (5 mL) was added to the clay film after irradiation, and a 1.0-mL aliquot of the supernatant obtained by centrifugation at 3000 rpm for 10 min was assayed spectrophotometrically in duplicate according to the method reported by Mottla et al. (1970). Hydrogen peroxide was quantified by using a standard response curve. The generation of singlet oxygen on the clay film was also examined according to the method reported by Gohre and Miller (1983). Kaolinite (1 g) was added to the pear-shaped Pyrex flask (20 mL) sealed up with a rubber septum, followed by an injection of DMF (5 rL). After irradiation for an appropriate period, methanol (6 mL) was added to quench the reaction and trans-diacetylethylene in the methanol extract was quantified by HPLC, using a standard response curve. RESULTS
Spectroscopy. The reflectance spectra of I adsorbed onto kaolinite and montmorillonite showed maxima at 275 and 283 nm, as shown in Figure 2. The UV absorption spectrum of I in 10% aqueous acetonitrile exhibited weak absorption at 275 (474.2 M-1 cm-l) and 282 nm (484.6 M-I cm-1) due to the n T* transition of the phenyl ring. There was no significant difference in the spectral patterns between the reflectance and absorption spectra of I. The IR spectra of I dispersed in KBr, kaolinite and KBr, and montmorillonite and KBr are shown in Figure 3. The strong absorption at 1400-930 cm-l was derived from the clays themselves. Although each absorption, especially peak(s) deriving from the P-S moiety observed at about 600-650 cm-1 (Hooge and Christen, 1958), could not be assigned, the shift caused by the clays was within 1cm-' in every case. On the basis of these spectra, it is likely
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J. Agric. FoodChem., Vol. 38,
photoinduced Oxidation of Tolclofos-methyl
therefore likely that the faster of the two reactions is due to photodegradation on the clay surfaces and the slower one due to a reaction in the interlayer space, which is largely independent of irradiation. The amounts of I and its degradation products formed under irradiation on the two clays are summarized in Table I. Under the influence of irradiation, the extractable 14C gradually decreased, and after 14 days, 45.2 3'% or 65.7 % of the activity originally applied was extracted from kaolinite or montmorillonite, respectively. The bound and the volatile activity correspondingly increased. On the kaolinite, bound activity was 35.1 % of that applied and on the montmorillonite 13.1% Corresponding figures for the volatile activity were 17.5% and 16.6%. The major components of the volatile 14C were I and 14C02with a trace of V . Since the amounts of bound 14C and volatile 14C in dark conditions were much smaller than those observed under irradiation, it may be inferred that most of the bound and volatile 14C is derived from the photoproducts, but not from the microbial degradation. The irradiation causes the gradual mineralization of I. It is likely that the photoproducts derived from the phenol moiety of I react with the active oxygen species followed by the degradation via the quinone intermediate finally to carbon dioxide (Crosby and Tutass, 1966; Choudhry, 1984). The main photoproduct in the extracts was 11, formed via oxidation of the P=S moiety of I, amounting to 9.9% and 42.8% a t 10 days on montmorillonite and kaolinite, respectively. Only a trace amount of I1 (0.4-2.8% a t 14 days) was detected in the dark control, which indicated that irradiation greatly enhanced the formation of 11. Although I and I1 were the only compounds detected under dark conditions, other photoproducts were identified on both clays after irradiation. I11 and I V formed via oxidation of the arylmethyl group and/or the P=S moiety and VI11 formed via rearrangement of the 0-methyl group to the sulfur atom of I were detected but amounted to less than 2.8% of the applied 14C. V-VII, formed via cleavage of the P-OAr bond followed by the successive oxidation of the arylmethyl group, were also detected but at less than 4.5% of the activity originally applied. Indirect Photolysis. The effects of molecular oxygen and water on the photodegradation of I are summarized in Table 11. The removal of water from montmorillonite and kaolinite retarded the photodegradation of I. The halflives of I on the dried clays were estimated to be 101.1 and 8.0 days for montmorillonite and kaolinite, respectively. The amount of I1 slightly decreased on the dry clays, as shown in Figure 5. The retarded photodegradation of I was also observed on kaolinite under N2 (half-life = 4.1 days). Since molecular oxygen and water are not considered to be completely removed from the clays under the conditions tested, the results obtained here are only qualitative. Furthermore, the E I - M S spectrum of I1 formed on kaolinite in the presence of H2'80 showed that the ratio of the peak intensities a t m / e 284,286, and 288 was 100135.1:60.2. This indicates that about 40% of the P=O oxygen is derived from 1'30. These results strongly suggest that both molecular oxygen and water participate in the photoinduced oxidation of the P=S moiety of I. Formation of Active Oxygen Species. The irradiation of t h e clays by themselves caused the formation of hydrogen peroxide (Table 111). The amount formed gradually increased with irradiation, reaching a maximum of 9.2 ppb after 8 h of irradiation on kaolinite. Hydrogen
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1400
1100
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Wavenumber (cm-')
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Figure 3. IR spectra of I. (a) KBr; (b)montmorillonite; (c) kaolinite.
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Figure 4. Decline curves of I. (0) Light, kaolinite; ( 0 )dark, light, montmorillonite; (m) dark, montmorillokaolinite; (0) nite. that the characteristic interactions of I with both of the clays are insignificant. Photolysis Studies. The decline curves of I are shown in Figure 4. Its degradation on the clay surface was significantly accelerated; the half-lives on kaolinite were estimated to be 2.1 days under irradiation compared with 115.2 days in the dark. In the case of montmorillonite, two reactions were involved under irradiation. The halflife of I in the slower reaction was 61.3 days on the basis of the data a t 7-14 days, which was fairly close to the halflife observed on montmorillonite in the dark of 54.1 days. The half-life of I in the faster reaction was calculated on the basis of t h e d a t a at 0-4 days t o be 1.5 days by subtracting the contribution from the slower reaction. This value was close t o t h a t observed for kaolinite under irradiation. According to Yaron (1978) montmorillonite adsorbs more organophosphorus pesticides into the interlayer space of the silicates than does kaolinite. It is
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1508 J. Agric. Food Chem., Vol. 38, No. 7, 1990
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Table 1. Amounts of I and Its Degradation Products on the Clay Films % of the applied 14C montmorillonite light dark 3 days 7 days 14days 7 days 14 days 3days 9.3 16.6 5.0 8.0 volatile 14C4 4.0 4.3 0.9 3.7 6.4 2.4 5.4 EG trapb 1.4 I V
0.9
